There are five major classes of proteases, categorized based on their mechanism of catalysis. These are the serine, cysteine, aspartic, threonine and metallo-proteases. Members of the cysteine protease class include deubiquitylation enzymes, caspases, calpains, cathepsins, viral, bacterial, protozoan or parasitic cysteine proteases.
Many proteins in the cell are regulated by the ubiquitin-proteasomal pathway (A. Hershko et al., Annu. Rev. Biochem., Annu Rev Biochem, 67: 425-79 (1998)). The conjugation and deconjugation of ubiquitin (Ub), a small (76 amino acids; 8.6 kDa) polypeptide, to proteins is mediated by families of enzymes (A. Hershko et al., supra). Typically, polyubiquitylated proteins are degraded by the proteasome.
Ubiquitin deconjugation is mediated by deubiquitylating enzymes (DUBs also known as deubiquitinating enzymes) that cleave the peptide bond of ubiquitin fused at its C-terminus to other proteins (including ubiquitin) or small molecules such as amines or thiols. In particular, DUBs may serve to spare certain proteins, or at least prolong their cellular lifetime, by removing the polyubiquitin tag, thereby preventing proteasomal degradation. DUBs are typically cysteine proteases and can be grouped into multiple classes. The largest class of DUBs, the ubiquitin specific protease (USP) family, cleave ubiquitin from a wide range of protein substrates (D. Komander et al., Nat. Rev. Mol. Cell. Biol., 10(8): 550-63 (2009); S. M. Nijman et al., Cell, 123(5): 773-86 (2005)). DUBs have been linked genetically and/or biochemically to a variety of diseases, including cancer, inflammatory disease, neurodegenerative disease, muscle wasting, infertility, and viral infection.
DUBs have gained interest as targets for the treatment of cancer. Attention was brought to the proteaseome pathway as a target for cancer therapy with the approval of the proteasome inhibitor bortezomib (Velcade®) for the treatment of multiple myeloma. Extended treatment with bortezomib is limited by its associated toxicity and drug resistance. However, therapeutic strategies that target specific aspects of the ubiquitin-proteasome pathway upstream of the proteasome, such as DUBs, are predicted to be better tolerated.
The DUB USP7 plays a key role in regulating the ubiquitylation of the RING-finger E3 ligase Mdm2 (and its human homolog Hdm2) (M. Li et al., Mol. Cell, 13(6): 879-86 (2004); J. Cummins et al., Nature, 428(6982): 1 p following 486 (2004)). Hdm2 binds the tumor suppressor, p53 and facilitates its degradation by the proteasome by polyubiquitylating p53 (R. Honda et al., FEBS Lett, 420(1): 25-7 (1997); Y. Haput et al., 387(6630): 296-9 (1997)). Importantly, a recent publication illustrates the therapeutic relevance of directly inhibiting USP7 catalytic activity as opposed to blocking the protein-protein interaction with its substrate (C. L. Brooks et al., Oncogene, 26(51): 7262-6 (2007).
In addition to the proven link between USP7, Hdm2 and p53, inhibition of USP7 is predicted to impact cancer by a variety of other mechanisms. For example, Hdm2 also binds to and inhibits the p53 paralog, p73 (E. Balint, Oncogene, 18(27): 3923-9 (1999)). Additional reported substrates of USP7 include claspin (H. Faustrup et al., Cell Biol., 184(1): 13-9 (2009)), the anti-proliferative protein PTEN (M. S. Song et al., Nature, 455(7214): 813-7 (2008)) and the anti-proliferative transcription factor, FOXO4 (A. van der Horst et al., Nat. Cell Biol., 2006. 8(10): 1064-73 (2006)).
Combination therapy approaches consisting of two or more therapeutic agents are widely used to treat many diseases including neoplastic diseases. When used effectively, combination therapies often exhibit synergistic therapeutic effects resulting in a reduction in dose and toxicity of the therapeutic agents.
Genotoxic agents such as Doxorubicin, Etoposide or Chlormethine are approved chemotherapeutic agents that damage tumor cell DNA by a variety of mechanisms resulting in tumor cell death (D. E. Thurston et al., Chemistry and Pharmacology of Anticancer Drugs, CRC Press, 37-84 (2007)). The USP7 substrate claspin is required for G2/M cell cycle arrest following DNA damage and inhibition of USP7 is predicted to prevent activation of the checkpoint kinase Chk1 and allow tumor cells to continue to proliferate in the presence of genotoxic agents. The continued proliferation of tumor cells in the presence of genotoxic agents results in tumor cell death. Thus a combination therapy utilizing a USP7 inhibitor with a genotoxic agent is predicted to result in synergistic tumor cell death.
U.S. Pat. Nos. 4,451,660 and 4,649,154, EP 31173, GB1459571, GB 1577743, US2007/0212712, US2006/0217381, JP03133975 and JP 04021677 disclose compounds comprising nitrothiophene. Nitrothiophene derivatives also were reported by Khimicheskaya Tekhnologiya, 50: 93-96 (2007), Journal of Chemical Research, Synopses, 7: 266-267 (2001), Journal of the Chemical Society, Perkin Transactions 2: Physical Organic Chemistry, 2: 325-334 (1998), Journal of the Chemical Society, Perkin Transactions 2: Physical Organic Chemistry, 2, 388-92 (1981). U.S. Pat. No. 4,994,485, WO 2005/070916 A1 and WO 2005/066152 A1 relate to cyanothiophene derivatives. Insofar as is known, however, it has not previously been disclosed nor suggested that nitrothiophene or cyanothiophene derivatives are effective for inhibiting deubiquitinating enzymes.
A number of inhibitors of USP7 have been reported. These include tetracyclic compounds (US2008/0103149 A1) and cyano-pyrazine derivatives (US2007/0032499 A1). See also WO 2004/009023 and F. Colland et al., Mol. Cancer. Ther., 8: 2286-95 (2009). However, no clinically approved compounds have emerged from these leads. Thus, clinically effective inhibitors of USP7 would fulfill a therapeutic need.
To date, there have been no reports of DUB inhibitors or activators that have successfully entered the clinic. One of the primary hurdles in the discovery and development of compounds that modulate DUB activity is the lack of a reliable, robust, high-throughput assay for the screening for inhibitors or activators of USPs. Progenra, the assignee of this application, has developed such an assay (U.S. Provisional Application No. 61/083,756). By utilizing its novel assay, Progenra has succeeded in identifying small molecule inhibitors of USP7.